High prevalence and co-infection of high-risk Human Papillomavirus genotypes among unvaccinated young women from Paraguay

Paraguay launched a human papillomavirus (HPV) vaccination program in 2013, so virological surveillance is important for measuring the impact of HPV vaccines. This study aimed to estimate the type-specific HPV frequency in unvaccinated sexually active women aged 18–25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination program. This study included 208 women, attending the Central Laboratory of Public Health between May 2020 and December 2021, were invited for testing through social networks and flyers at local health centers and higher education institutes. All participants who agreed to contribute to the study signed a free, prior, and informed consent form and answered a questionnaire that included basic demographic data and determining factors of HPV infection. Human papillomavirus detection and genotyping were conducted using the CLART HPV2 test (Genomica, Madrid, Spain) that allows the individual identification of 35 genotypes. 54.8% women were positive for any HPV type, with 42.3% positive for high-risk HPV (HR-HPV) types. Several factors were associated with HPV detection including the number of sexual partners, new sexual partners, non-use of condoms, and history of other sexual infections. Moreover, multiple infections were identified in 43.0% of the young women. We detected 29 different viral types present in both single and multiple infections. HPV-58 was the most commonly detected HPV type (14.9%), followed by HPV-16, HPV-51, and HPV-66 (12.3%). We estimated the prevalence of bivalent (16/18), quadrivalent (6/11/16/18), and nonavalent (6/11/16/18/31/33/45/52/58) vaccine types to be 8.2%, 13%, and 38%, respectively. These results reinforce the importance of surveillance studies and provide the first data regarding circulating HPV genotypes in the unvaccinated population in Paraguay, thus generating a baseline to compare future changes in the overall and type-specific HPV prevalence after HPV vaccination.

Introduction immunotherapy) [11]. Minimally invasive surgery has been studied and associated with advantages in the treatment of various gynecological cancers [13].
In Paraguay, the quadrivalent HPV vaccine was introduced into the national immunization program in 2013, prioritizing girls ages 9-10 years old with 0-1-6-month immunization schedule using school-based vaccination [14].
The detection of HPV infection can precede advanced stages of the disease, such as cervical intraepithelial neoplasia grade 3 (CIN 3), for a period of five to ten years; therefore, identifying prevalent HPV genotypes can provide earlier clues about the impact of the vaccine program [9,15].
In Paraguay, primary screening aims to detect precursor lesions of cervical cancer in women over 30 years of age [14], making screening data inadequate for assessing the early impact of the vaccine. Consequently, establishing a national HPV surveillance program that includes a series of cross-sectional studies to assess the prevalence of HPV in cervical samples from young women would aid in identifying the impact of the vaccination program [9].
This study aimed to estimate the type-specific HPV frequency in unvaccinated women aged 18-25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination program. Here, we provide the first data about circulating genotypes in the unvaccinated population, generating a baseline to compare future changes in overall and type-specific HPV prevalence after HPV vaccination.

Study population
This cross-sectional study was conducted between May 2020 and December 2021, using convenience sampling strategy, due to the national primary screening is mainly aimed at women aged 30 years and above [14]. Sexually active women aged 18-25 years, who had not been vaccinated against HPV were invited for screening through social networks and flyers at local health centers and higher education institutes. Exclusion criteria were pregnancy, nonsexual onset at the time of the study and have been received HPV vaccine. Women who voluntary agreed to participate signed a free, prior, and informed consent form and answered a questionnaire that included basic demographic data and determining factors of HPV infection (age, origin, occupation, educational degree, age at first sexual intercourse, number of sexual partners in the last 12 months, contraceptive methods, sexual habits, alcohol consumption, and tobacco use). 208 young women were included, and their samples were processed at the Central Laboratory of Public Health (LCSP), the National Reference HPV Laboratory in Paraguay.

Clinical specimen collection
After a personal interview, specimens for HPV detection were collected from the endocervix/ ectocervix using a cervical cytology brush (PAPETTE1 Cervical Cell Collector, WALLACH Surgical Devices, Trumbull, CT, USA). The samples were resuspended by shaking the brush in vials with 2.0 ml of sterile phosphate-buffered saline (PBS) to detach the collected cells. The brush was discarded, and the samples were kept at 8˚C until processing within 72 h. All samples were assigned an anonymous and unique patient code.

DNA extraction from cervical samples
Aliquots of 1.0 ml were centrifuged at 12,000 rpm for 10 min to pellet the exfoliated cervical cells. After removing the supernatant, 1.0 ml of sterile water was added to resuspend and wash the cells. The samples were centrifuged again at 12,000 rpm for 10 min. The supernatants were discarded, and the pellets were resuspended in a mix of 180 μl PBS and 20 μl proteinase K (20 μg/ml). The samples were vortexed and incubated for 1 h at 65˚C. Finally, HPV DNA was extracted using a CLART HPV 2 DNA Extraction/Purification Kit (Genomica, Madrid, Spain) according to the manufacturer's instructions, and 100 μl of eluted DNA was stored at -20˚C until use.
We used 5 μl of purified DNA for PCR amplification and the PCR products were labeled with biotin. The PCR products were denatured at 95˚C for 10 min and visualized using 5 μl of the denatured PCR products. Hybridization between amplicons and their specific probes was performed using probes immobilized in the wells of the hybridization plate. A streptavidin conjugate was added to the wells, which bound to the biotin-labeled PCR products, resulting in the formation of an insoluble peroxidase precipitate. The precipitate was analyzed using a Clinical Array Reader (Genomica, Madrid, Spain). Samples with invalid outcomes were retested, and the second result was considered conclusive.

Statistical analyses
A descriptive analysis of the epidemiological characteristics of study participants was performed. All variables were recorded and coded in Microsoft excel 2013 and data were processed using the Epi Info 7 version 7.2.2.16 (EPI INFO by CDC, USA). Participants were counted more than once for assessing the prevalence of HR-HPV, probable/possible HR-HPV, and LR-HPV if they had types from more than one group. Single infection was defined as infection with one HPV type and multiple HPV infections were defined as the detection of two or more HPV types in the same sample. Frequencies and percentages were determined for the participant characteristics. The distribution of positive cases and genotypes was analyzed. The chi-square test was used to compare proportions. Odd ratios (OR) with 95% confidence interval were calculated to evaluate the association between HPV infection and sociodemographic and behavioral characteristics. For all data analyses and relationships between variables, a value of p<0.05 was considered statistically significant.

Ethical considerations
The LCSP Ethics Committee reviewed and approved the study (resolution N˚110/2019). The participants signed a free and informed written consent form before inclusion in the study. No women younger than 18 years old were included in the study. Since in Paraguay, primary screening was aimed mainly at women aged 30 years and above, to detect precursor lesions of cervical cancer; special care was taken to explain the scope of this research study to the participants. The HPV tests results were communicated to the patients by trained health personnel with an extensive health education to inform women and avoid unnecessary additional anxiety and distress, helping to understand HPV and its association with cervical cancer, as well as the importance of screening.

Results
The average age of study participants was 22.5±2.1 years. Of these, 70.2% had university as education level. The mean age of first sexual intercourse was 16.8±2.1 years, and 33.2% reported a previous history of STI. Additionally, 59.1% of the participants reported having fewer than two sexual partners in the last year, 81.7% had no history of smoking, and 72.6% reported alcohol consumption (Table 1). A total of 208 cervical samples were analyzed, all of which yielded a positive result for the human CFTR gene, proving the DNA integrity and good sample quality. HPV DNA was detected in 114 specimens, resulting in an overall prevalence of 54.8% (95% CI 48.0-61.6).
The results were further analyzed for cross-sectional associations between HPV infection and sociodemographic, obstetric, and behavioral characteristics. We observed significant associations between HPV infection and women who reported not living with their partner (OR 3.25; 95% CI 1.53-6.88; p = 0.01), women with more than one sexual partner within the past  (Fig 1).

Discussion
The WHO global strategy for the elimination of cervical cancer as a public health problem proposes a multidisciplinary approach that includes community education, social mobilization, vaccination, detection, treatment, and palliative care.
HPV vaccination in combination with timely detection and appropriate treatment of cervical cancer precursor lesions is considered to have the potential to achieve the elimination of this disease, defined by the WHO as fewer than 4 new cases per 100,000 women-years [8].
With the introduction of the HPV vaccine, changes in the transmission dynamics of the virus are expected, making necessary to monitor changes in the prevalence of HPV and the patterns of HPV-associated diseases. Population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low-and middle-income countries, so cross-sectional surveys in sentinel populations could be a suitable alternative [8].
Paraguay, like many other countries worldwide, has incorporated the HPV vaccine into its national immunization program, requiring a virological surveillance to measure its impact. It can provide an early measure of vaccine effectiveness by quantifying the magnitude of reduction in HPV type-specific prevalence, including possible evidence of herd and cross protection and provide timely assessment of vaccination strategy. The principle of HPV prevalence surveillance is to recruit samples in age groups at risk of HPV exposure and conduct repeat-cross sectional analyses [16].

PLOS ONE
In this study, we analyzed a cohort of unvaccinated sexually active women between 18-25 years of age and found an overall HPV prevalence of 54.8%, comparable to others cross-sectional studies in South American as Colombia with a prevalence of 60.3% in a same age group [17], and Argentina with a prevalence of 56.3% in 15 to 17-year-old female adolescents [18], showing the high of HPV infections in young women in the region. Our observed HPV prevalence rate is also like the 55% infection rate observed at enrollment in a longitudinal study of young women [19]. However, this rate was higher than that observed in studies in unvaccinated young women from United States (47.5% for 21-24 years) [20] and Europe, which has ranged from 25% to 33% among women aged <25 years [21-23]. Thus, the overall HPV prevalence is low in developed countries [24].
Previous studies have established sexual contact as the main route of HPV transmission. Thus, the higher HPV prevalence in women �25 years of age could be explained by recent first sexual contact, a condition that increases the chance of exposure to the virus [5,24,25].
Although the demographic data and all risk factors for acquiring HPV showed that the participants were moderate risk e.g., only 24.0% used condom, 33% reported STI, 40.9% had reported 2 or more partners, the results reported high risk HPV types. At least one type of HR-HPV was found in 42.3% of young women, with HR-HPV prevalence ranging from 18.7%-45.7% among women under 25 years old worldwide [26][27][28][29][30][31][32][33]. A similar rate (42.2%) has  As expected, sexual behavior is associated with an increased risk of HPV infection, as well as the number of sexual partners, new sexual partners, non-use of condoms, and a history of other sexual infections. A high number of sexual partners is the most important risk factor for HPV positivity, and newly acquired sexual partners further increase this risk [5,25,36]. Condom use may help reduce the risk of cervical and vulvovaginal HPV infection [37]. Other STI like Chlamydia trachomatis have been associated with cell inflammatory processes in the cervix, facilitating HPV entry and reducing its elimination [25,38].
We identified multiple infections in 43.0% of all cases, as previously reported in younger groups of women [39,40]. A few studies have associated multiple HPV infections with an increased risk of cervical intraepithelial neoplasia [41,42]. However, other studies found no significant difference in the development of cervical intraepithelial neoplasia and cancer in women with single or multiple infections [43,44]. The total risk of precancer in women infected with several HPV types was increased compared to women infected with a single HPV type [45]. It is not clear whether the risk from multiple infections is greater than the sum of the risks posed by individual HPV types. However, despite the presence of multiple viral types in a sample, only one HR-HPV leads to potential malignant transformation [46].
It is established that persistent infections by HPV-16 have a higher probability of causing precancerous lesions than infection with other viral types [48,49]. Women with normal cytology and persistent HPV-16 infection have an approximately 30% chance of developing highgrade lesions in a 12-month period [50]. HPV-58 is also included in high-risk group and has been frequently reported in cervical cancer and high-grade lesions in several regions of the world [51], such as Asia [52,53], Mexico [54,55], Costa Rica [43], and Brazil [56]. Given the wide circulation of HPV-58 in the population studied and the evidence of its oncogenic potential, particular attention should be given to HPV-58 infection.
We found that 8.2% of the women were infected with HPV-16/18 that are included in the bivalent vaccine, close to that reported in similar populations from Colombia (12.9%) [17] and the United States (10.0%) [57]. Infections with types included in the quadrivalent vaccine (HPV-6/11/16/18) were detected in 13.0% of women, comparable to a nationally representative United States survey of females conducted from 2003 to 2006 with a prevalence of 18.5% in the 20-24-year-old group [58], but less than that reported in Argentine adolescents (22.5%) [18]. HPV 31, 33, and 45 were detected in 10.6% of women. These infections could also be prevented, as the bivalent and quadrivalent vaccines provide cross-protection against these genotypes [10].
HPV-58, not targeted by the vaccine currently in use in Paraguay's vaccination program was the most prevalent type in this studied group. Consequently, the incidence and risk of non-vaccine HPV types must be considered to assess the impact of the quadrivalent vaccine used in the country in the national immunization program.
Have been reported women immunized with quadrivalent vaccine at young age who still developed cervical intraepithelial neoplasia with evidence that these lesions are commonly associated with HPV types not targeted by this vaccine, but some of the HPV types associated with these lesions in vaccinated women are targeted by the Gardasil-9 [59].
As we know, the nonavalent vaccine contains five additional oncogenic types (HPV-31, 33, 45, 52, and 58) and according to the current data, this vaccine could prevent 38.0% of HPV infections identified in this study. Therefore, the high prevalence of HPV types targeted by nonavalent HPV vaccines could support its introduction in the national immunization program to achieve greater coverage, considering that the preventable proportion of cervical cancers is estimated to increase to 90% [60].
The present study had some limitations as the small sample size of population studied, for this reason the results cannot be extrapolated to the total young women from Paraguay. The recruitment strategy by social networks and flyers at local health centers and higher education institutes may have induced a selection bias. Although this was not a population-based study, the results are particularly important, providing information regarded HPV prevalence in groups that are not often included in epidemiological surveys or cervical cancer prevention programs. We know that the population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low resources countries, thus cross-sectional surveys in sentinel populations could be a suitable alternative.
For hence this study even its important limitations, such as the small sample size and the convenience recruitment strategy, the results are particularly important, providing information regarding HPV prevalence in groups that are no often included in national cervical cancer prevention programs. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the short-term estimation of the impact of the national HPV immunization program.
Even though several studies provide evidence that genital HPV infection is very common in young sexually active women and in most cases is a transient and self-limited infection, the evaluation of overall HPV prevalence in this population has been proposed as a short-term endpoint for determining the impact of HPV vaccination in surveillance programs.
This research could provide key information to healthcare policy makers about the importance of increasing HPV vaccination coverage in national immunization programs. The results should be combined with other studies in cohorts of vaccinated women to investigate the reduction of types 16 and 18, as well as their possible replacement by other genotypes, thus evaluating the impact of the implementation of HPV vaccination in Paraguay and compare the results with other countries.
In addition, these data can improve the acceptance of the HPV vaccine in the Paraguayan population, highlighting the high prevalence of HPV, especially the high-risk types, observed in unvaccinated young women. Increased public awareness of the benefits of HPV vaccination for cervical cancer prevention could increase public support for immunization.
In conclusion, this is the first report on the prevalence of HPV and its genotype distribution, including HPV types targeted by current vaccines, in unvaccinated young sexually active women from Paraguay. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the estimation of the impact of the national HPV immunization program. Surveillance and monitoring of changes in viral transmission that lead to modification of HPV type-specific prevalence and the patters of HPVassociated disease are crucial to progress toward the global elimination of cervical cancer.